WO2017156491A1 - Tlx et mir-219 utilisés comme cibles thérapeutiques potentielles pour des troubles neurodéveloppementaux - Google Patents

Tlx et mir-219 utilisés comme cibles thérapeutiques potentielles pour des troubles neurodéveloppementaux Download PDF

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WO2017156491A1
WO2017156491A1 PCT/US2017/021962 US2017021962W WO2017156491A1 WO 2017156491 A1 WO2017156491 A1 WO 2017156491A1 US 2017021962 W US2017021962 W US 2017021962W WO 2017156491 A1 WO2017156491 A1 WO 2017156491A1
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mir
tlx
nscs
inhibitor
cells
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Yanhong Shi
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City Of Hope
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Definitions

  • TLX is a nuclear receptor that plays a critical role in vertebrate brain function [1 -3]. It is an essential regulator of adult neural stem cell (NSC) self-renewal [3-5] and plays an important role in enhancing learning and memory by regulating adult hippocampal neurogenesis [6,7]. It also plays a role in neurodevelopment through regulation of cell cycle progression in embryonic NSCs [4,8-10]. TLX is a well-characterized transcriptional regulator. It controls target gene expression partly by recruiting transcriptional corepressors, such as HDACs and LSD1 [1 1 -13].
  • TLX represses the transcription of GFAP, p21 , pten, and microRNAs miR-9 and miR-137, but activates Wnt signaling, SIRT1 and MASH1 in NSCs [3,5, 1 1 , 12, 14-16].
  • TLX represses the transcription of GFAP, p21 , pten, and microRNAs miR-9 and miR-137, but activates Wnt signaling, SIRT1 and MASH1 in NSCs [3,5, 1 1 , 12, 14-16].
  • TLX represses the transcription of GFAP, p21 , pten, and microRNAs miR-9 and miR-137, but activates Wnt signaling, SIRT1 and MASH1 in NSCs [3,5, 1 1 , 12, 14-16].
  • miRNAs are small non-coding RNAs that regulate gene expression through translational inhibition or RNA degradation [17].
  • the biogenesis of miRNAs starts from primary transcripts (pri-miRNAs), which are processed by the nuclear RNasell l Drosha into precursor miRNAs (pre-miRNAs) that contain hairpin loop structures.
  • pri-miRNAs primary transcripts
  • pre-miRNAs precursor miRNAs
  • Mature miRNAs are incorporated into an RNA-induced silencing complex (RISC) to repress target mRNAs.
  • RISC RNA-induced silencing complex
  • the Drosha complex consists of Drosha, DiGeorge syndrome critical gene 8 (DGCR8), RNA helicase p68 (DDX5) and p72 (DDX17) [18-20].
  • DGCR8 DiGeorge syndrome critical gene 8
  • DDX5 RNA helicase p68
  • p72 DDX17
  • miR-219 is an miRNA that is specifically expressed in the brain [24,25]. It promotes oligodendrocyte differentiation by repressing negative regulators of oligodendrocyte differentiation [26,27]. In a recent study, miR-219 was shown to promote neural precursor cell differentiation in zebrafish by inhibiting apical polarity proteins, par-3 family cell polarity regulator (PARD) and protein kinase C iota (PRKCI) [28]. However, whether miR-219 regulates the phenotypes of neural stem/progenitor cells (collectively referred to as NSCs) in mammalian brains remains unknown.
  • NSCs neural stem/progenitor cells
  • this disclosure relates to a method for treating a neurodevelopmental disorder in a subject.
  • the method entails inhibiting, repressing, or down-regulating expression of miR-219, overexpressing or up-regulating the expression of TLX, or a combination thereof.
  • the expression of miR-219 is inhibited, repressed or down-regulated by administering to the subject a therapeutically effective amount of one or more antagonists of miR-219.
  • expression of TLX is up-regulated by administering to the subject a therapeutically effective amount of one or more TLX agonists or a vector expressing a gene encoding TLX.
  • Figures 1 a-1 g illustrate that TLX inhibits miR-219 processing in NSCs.
  • Figure 1 a shows elevated expression of mature miR-219 in TLX KO mouse brains, compared to WT mouse brains, revealed by Northern blot analysis. U6 is included as a loading control.
  • Figure 1 b shows the levels of the two primary forms of miR-219, pri-miR-219-1 and pri-miR-219-2, exhibited minimal change in WT and TLX KO mouse brains, as analyzed by RT-PCR.
  • Figure 1 d shows the levels of pre-miR-219 and mature miR-219, but not pri-miR-219, increased significantly in TLX knockdown NSCs independent of actinomycin D treatment.
  • siC control RNA
  • siTLX TLX siRNA.
  • n 6.
  • Figure 1 e shows a scheme for monitoring miRNA processing using a luciferase reporter. The miRNA processing activity is inversely correlated with the luciferase activity.
  • Figure 1f shows overexpression of TLX inhibits miR-219 processing from pri-miR-219 to pre-miR-219. miR-1224-Glo was included as a negative control.
  • n represents experimental repeats in panels c, d, f & g. Error bars are sd of the mean for all the quantification in this study. For each representative image, the experiments were repeated three times or more.
  • Figures 2a-2f illustrate that TLX interacts with the miRNA processing machinery.
  • Figure 2a shows a scheme for identifying TLX-interacting proteins using mass spectrometry (MS) analysis.
  • Figure 2b shows differentially represented proteins in the HA immunoprecipitates of control HA or HA-TLX-expressing HeLa cells. Arrow indicates a protein band of 68 kD that is specifically detected in the HA immunoprecipitates of HA-TLX-expressing HeLa cells.
  • Figure 2c shows interaction of TLX with p68, Drosha and DGCR8. Lysates of HA-TLX transfected HEK293T cells were treated with or without DNase and RNase, then immunoprecipitated with HA antibody or IgG control.
  • the immunoprecipitates were blotted with p68 antibody.
  • lysates of Flag-Drosha and HA-TLX or Flag-DGCR8 and HA-TLX co- transfected HEK293T cells were treated with or without DNase and RNase.
  • Cell lysates were immunoprecipitated with anti-Flag antibody, then blotted with anti-HA antibody.
  • Figure 2d shows interaction of TLX with Drosha and DGCR8 in mouse brains. Lysates of embryonic mouse brains were immunoprecipitated with TLX antibody, then blotted with anti-Drosha, anti-DGCR8 or anti-TLX antibody.
  • Figure 2e shows a scheme for RNA immunoprecipitation.
  • Lysates of NSCs transduced with TLX siRNA were immunoprecipitated with anti-Drosha, anti-DGCR8, or anti-TLX antibody. RNAs were extracted from the immunoprecipitates, and subjected to RT- PCR for pri-miR-219. Figure 2f shows TLX knockdown promoted the binding of Drosha and DGCR8 to pri-miR-219. Lysates of NSCs transduced with siC or siTLX were immunoprecipitated with IgG control or indicated antibodies. pri-miR-219 RNA associated with Drosha (indicated by solid arrows) or DGCR8 (indicated by open arrows) was determined by RT-PCR.
  • Figures 3a-3j illustrate that miR-219 inhibits NSC proliferation & promotes neuronal differentiation.
  • Figures 3a and 3b show overexpressing miR-219 in NSCs inhibited cell proliferation (Figure 3a) and promoted neuronal differentiation (Figure 3b).
  • BrdU or Tujl staining is shown in red and Dapi counterstaining is shown in blue.
  • Figures 3c and 3d show quantification of BrdU+ cells (Figure 3c) and Tuj1 + cells (Figure 3d) in control RNA (C) and miR-219-treated NSCs.
  • n 5, *p ⁇ 0.001 by student's t-test for both panels.
  • N represents experimental repeats.
  • Figure 3e illustrate that miR-219 inhibits NSC proliferation & promotes neuronal differentiation.
  • Figures 3a and 3b show overexpressing miR-219 in NSCs inhibited cell proliferation (Figure 3a) and promoted neuronal differentiation (Figure 3b).
  • FIG. 1 shows in utero electroporation of miR-219 decreased NSC proliferation in the VZ/SVZ of embryonic brains. Electroporated cells were labeled by RFP and proliferating cells were labeled by Ki67.
  • Figure 3g shows electroporation of miR-219 induced precocious outward cell migration. The electroporated brains were stained for neuronal marker doublecortin (DCX). Transfected cells were labeled by RFP.
  • DCX neuronal marker doublecortin
  • Figure 3i shows higher magnification images of RFP+DCX+ cells at the CP of brains electroporated with control RNA or miR-219. Scale bar: 50 ⁇ for panels a, b, & e; 100 ⁇ for panel g; 25 ⁇ for panel i.
  • Figures 4a-4d illustrate that an miR-219 inhibitor reversed NSC phenotypes induced by TLX siRNA in vivo.
  • Figure 4a shows co-electroporation of TLX siRNA with an miR-219 inhibitor rescued the decrease in NSC proliferation induced by TLX siRNA.
  • E13.5 mouse brains were electroporated in utero with 1 ) a control RNA and the RFP reporter (siC-RFP), 2) TLX siRNA and the RFP reporter (siTLX-RFP), 3) an miR-219 inhibitor with siC-RFP, or 4) an miR-219 inhibitor with siTLX-RFP.
  • the electroporated cells were labeled by RFP and proliferating cells were labeled by Ki67.
  • Figure 4c shows electroporation was performed as described in panel e and brain sections were stained for neuronal marker DCX. Migration of the electroporated cells was tracked by RFP fluorescence.
  • Figures 5a-5i illustrate that a TLX peptide promotes miR-219 processing.
  • Figure 5a shows mapping p68 and Drosha-interacting domain in TLX.
  • a schematic of TLX deletion mutants and the Drosha/p68 interacting domain (Dpi) is shown on the left.
  • a summary of p68 and Drosha binding results is shown on the right.
  • Figure 5b shows deletion of TLX residues 340 to 359 reduced the interaction of TLX with p68 substantially.
  • HEK293T cells were transfected with HA-tagged full length TLX (residues 1 -385) or its deletion mutants (residues 1 -306, 1 -340, or 1 -359).
  • Lysates were immunoprecipitated (IP) with HA antibody (aHA), then probed with p68 antibody ( ⁇ 68) in Western blot analysis (WB).
  • Figure 5c shows deletion of TLX residues 340 to 359 reduced TLX interaction with Drosha.
  • HEK293T cells were transfected with Flag-tagged Drosha and HA-tagged full length or deletion mutants of TLX.
  • Lysates were IP with Flag antibody (aFlag), then probed with HA antibody (aHA). A non-specific (ns) band in the Western blot was indicated.
  • Figures 5d and 5e show expressing the Dpi peptide abolished the interaction of TLX with Drosha ( Figure 5d), but not the interaction of TLX with HDAC5 ( Figure 5e), as revealed by co-IP analysis.
  • An empty vector (-) and a control peptide (C) were included as negative controls for the Dpi peptide.
  • Cell lysates were IP with anti-Flag antibody, then blotted with anti-HA or anti-Flag antibody. The expression of individual proteins in the transfected cells was shown by immunoblotting as input.
  • Figure 5f shows expression of the Dpi peptide promotes miR-219 processing. miR-219 processing was monitored using the miR-219-Glo reporter.
  • Figures 6a-6g illustrate that the Dpi peptide regulates NSC proliferation and differentiation.
  • Figures 6a-6c show expression of the Dpi peptide inhibits NSC proliferation and promotes neuronal differentiation, and this effect could be reversed by the miR-219 inhibitor, TuD-miR-219.
  • Mouse embryonic NSCs were transduced with virus expressing the Dpi peptide or a control peptide (C), in the absence or presence of TuD-miR-219. The virus transduced cells were labeled by a GFP reporter.
  • E13.5 mouse brains were electroporated in utero with vectors expressing: 1 ) a control peptide and RFP reporter (C); 2) Dpi peptide and RFP reporter (Dpi); 3) TuD-miR-219 plus control peptide and RFP reporter (TuD- miR-219+C); or 4) TuD-miR-219 plus Dpi and RFP reporter (TuD-miR-219 + Dpi).
  • the electroporated cells were labeled by RFP, proliferating cells were labeled by Ki67 ( Figure 6e), and neuronal cells were labeled by DCX ( Figure 6f).
  • Figures 7a-7f illustrate that the DISC1 -mutant NSCs exhibit increased miR-219 expression and reduced proliferation.
  • Figure 7a shows a schematic diagram showing the pedigree for iPSC generation. iPSCs from a wild type (WT) individual outside of the pedigree (C1 ) were included as a control. The + and - signs represent the presence and absence of the 4 bp deletion in the DISC1 gene, respectively. The squares represent male, while the circles represent female.
  • Figure 7b shows NSCs derived from both WT (C1 , C2, & C3) and DISC1 -mutant iPSCs (D1 , D2, C1 M and C3M) expressed neural precursor markers SOX1 and NESTIN. Scale bar: 50 ⁇ .
  • Figures 7c and 7d show RT-PCR showing elevated expression of miR- 219 (Figure 7c) and reduced expression of TLX ( Figure 7d) in DISC-mutant NSCs (D1 , D2, C1 M and C3M), compared to that in WT NSCs (C1 , C2, & C3).
  • Figures 7e and 7f show the DISC1 -mutant NSCs (D1 , D2, C1 M and C3M) exhibited reduced cell proliferation (Figure 7e) and precocious neuronal differentiation (Figure 7f).
  • NSC proliferation rate was determined by the percentage of BrdU+SOX1 + cells.
  • Neuronal differentiation rate was determined by the percentage of Tuj1 + cells.
  • n 4 for panels c-f.
  • N represents experimental repeats. ANOVA test result was shown below each graph.
  • Figures 8a-8d illustrate that inhibition of miR-219 or overexpression of TLX rescues reduced cell proliferation in SCZ NSCs.
  • Figure 8a shows overexpression of miR-219 inhibited cell proliferation in WT NSCs.
  • WT C1 , C2, C3
  • DISC1 -mutant NSCs D1 , D2, C1 M, C3M
  • NSC proliferation rate was determined by the percentage of BrdU+SOX1 + cells.
  • Figure 8b shows TuD-miR-219 rescued the proliferative defect in DISC1 mutant NSCs.
  • WT and DISC1 -mutant NSCs were transduced with a control vector (-miR- 219-TuD) or TuD-miR-219-expressing vector (+miR-219-TuD).
  • NSC proliferation rate was determined by the percentage of BrdU+SOX1 + cells.
  • Figure 8c shows knockdown of TLX inhibited cell proliferation in WT NSCs.
  • WT (C1 , C3) and DISCI - mutant NSCs (D1 , D2, C3M) were transduced with virus expressing a control RNA (- siTLX) or TLX siRNA (+siTLX).
  • NSC proliferation rate was determined by the percentage of BrdU+SOX1 + cells.
  • Figure 8d shows overexpression of TLX rescued the proliferative defect in DISC1 mutant NSCs.
  • WT and DISC1 -mutant NSCs were transduced with control vector (-TLX) or TLX-expressing vector (+TLX).
  • Figure 9 shows the knockdown of TLX expression in NSCs.
  • Mouse NSCs were transduced with lentivirus expressing a scrambled control RNA (siC) or TLX siRNA (siTLX).
  • Figures 10a-10c illustrate the lack of toxicity and gliogenic induction in miR-219-transfected cells.
  • Figures 10b and 10c show no induction of GFAP and MBP expression in miR-219- electroporated mouse brains at E15.5. Scale bar: 100 ⁇ .
  • Figures 1 1 a-1 1 g illustrate that TLX-miR-219 regulates the expression of PDGFRa in NSCs.
  • Figure 1 1 a shows PDGFRa expression was reduced in TLX KO mouse brains. The expression of PDGFRa in WT and TLX KO mouse brains was examined by RT-PCR. GAPDH was included as a loading control.
  • Figures 1 1 c-1 1 e show the expression pattern of miR-219 in NSCs and neurons inversely correlates with that of PDGFRa and TLX.
  • Figure 1 1 g shows inhibition of PDGFRa expression by TLX siRNA could be rescued by the miR-219 decoy inhibitor, TuD-miR-219.
  • the expression of PDGFRa in NSCs transduced with scramble control RNA (siC) or TLX siRNA (si-TLX), in the absence or presence of TuD-miR- 219, was examined by RT-PCR. n 5. *p ⁇ 0.01 by student's t test.
  • Figures 12a-12d illustrate that knockdown of PDGFRa inhibits NSC proliferation & promotes neuronal differentiation and migration in embryonic mouse brains.
  • Figure 12a shows electroporation of PDGFRa siRNA decreased NSC proliferation in the VZ/SVZ of embryonic mouse brains. The electroporated cells were labeled with RFP and proliferating cells were labeled with Ki67.
  • Figure 12c shows electroporation of PDGFRa siRNA led to precocious outward cell migration.
  • the electroporated brains were labeled by RFP and stained for the neuronal marker doublecortin (DCX).
  • Figures 13a-13d illustrate that PDGFRa functions downstream of miR- 219 in embryonic mouse brains.
  • Figure 13a shows co-electroporation with PDGFRa and miR-219 reversed the decrease in NSC proliferation induced by miR-219 in the VZ/SVZ of embryonic mouse brains. The electroporated cells were labeled with RFP and proliferating cells were labeled with Ki67.
  • Figure 13b shows co-electroporation with PDGFRa and miR-219 reversed precocious outward cell migration induced by electroporation with miR-219 alone.
  • Figures 13c and 13d show the percentage of RFP+KJ67+ cells (Figure 13c) or cells migrated to the CP ( Figure 13d) out of total RFP+ cells in miR-219 or miR-219 and PDGFRa-electroporated brains is shown.
  • n 3 for panels c & d.
  • Figures 14a-14d illustrate that PDGFRa functions downstream of TLX in embryonic mouse brains.
  • Figure 14a shows co-electroporation with PDGFRa and TLX siRNA (siTLX + PDGFRa) reversed the decrease in NSC proliferation in the VZ/SVZ induced by TLX siRNA alone.
  • a control RNA (siC) was included as a negative control for TLX siRNA.
  • Figure 14c shows co-electroporation with PDGFRa and TLX siRNA reversed the outward cell migration induced by TLX siRNA alone. The electroporated cells were labeled by RFP.
  • Figures 15a-15b show that miR-219 regulates neuronal differentiation in SCZ NSCs.
  • Figure 15a shows overexpression of miR-219 promotes neuronal differentiation in WT NSCs.
  • WT C1 , C2, C3
  • DISC1 -mutant NSCs D1 , D2, C1 M, C3M
  • Neuronal differentiation rate was determined by the percentage of Tuj1 + cells.
  • Figure 15b shows inhibition of miR-219 reverses precocious neuronal differentiation in SCZ NSCs.
  • Figure 16 shows the list of Northern blot probes and RT-PCR primers (SEQ ID NOS: 1 -34). DETAILED DESCRIPTION
  • a method of correcting a defective rate of proliferation in a population of neural stem cells is provided.
  • This embodiment may include a step of contacting the population of NSCs with an effective amount of an miR-219 inhibitor, an agent to increase expression or activity of TLX, or both.
  • the miR-219 inhibitor used in the embodiments described herein may be any suitable agent that inhibits the expression or activity of miR-219 including, but not limited to, a tough decoy RNA, an RNAi molecule (e.g., shRNA, siRNA, or any other RNA interference molecule), or an aptamer.
  • the miR- 219 inhibitor is an miR-219-5p hairpin inhibitor (Dharmacon) or TuD-miR-219 (a tough decoy RNA).
  • TuD-miR-219 has the following sequence (SEQ ID NO:35):
  • the agent to increase expression or activity of TLX may be any suitable agent including, but not limited to, an agent to chemically modify TLX or a vector expressing a gene encoding TLX.
  • the vector expressing TLX may be a plasmid or any suitable recombinant viral vector capable of delivering a nucleotide sequence that is expressed in a cell including, but not limited to, a lentiviral vector, an adenoviral vector, an AAV vector, or any other suitable recombinant viral vector.
  • the vectors described herein may be designed to include the nucleotide sequence of TLX, reproduced below: Human NR2E1 (TLX) nucleotide sequence (SEQ ID NO:36):
  • nucleotide sequence above is translated to express the TLX protein, the amino acid sequence of which is shown below:
  • a portion of TLX may be used as the agent to increase expression or activity of TLX, such as the Dpi domain of TLX.
  • the Dpi domain may be expressed by the vector or delivered to the population of NSCs.
  • the nucleotide and amino acid sequences of Dpi are shown below: Human TLX DPI nucleotide coding sequence (1021 to 1077nt) (SEQ ID NO:38):
  • the mir-219 inhibitor, the agent to increase expression or activity of TLX, or both may be used to contact the population of NSCs in vitro.
  • the population of in vitro cells are derived from a subject suffering from a neurodevelopmental disorder such as schizophrenia, bipolar disorder, or depression.
  • the agent to increase expression or activity of TLX, or both may be used to contact the population of NSCs in vivo.
  • the population of in vivo cells are present in the nervous system of a subject suffering from schizophrenia, bipolar disorder, or depression.
  • the mir-219 inhibitor, the agent to increase expression or activity of TLX, or both causes an increase in NSC proliferation rate, and can therefore be used in methods for treating neurodevelopmental disorders that are associated with having a defective proliferation rate of NSCs, such as schizophrenia, bipolar disorder, or depression.
  • the mir-219 inhibitor, the agent to increase expression or activity of TLX may be used separately or in combination to increase proliferation of NSCs in a subject suffering from one of these disorders.
  • treating refers to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
  • treating a condition means that the condition is cured without recurrence.
  • a therapeutically effective amount refers to an amount of an agent, including a nucleic acid, a peptide, or a chemical compound, or a composition that produces a desired therapeutic effect.
  • the precise therapeutically effective amount is an amount of the agent or composition that will yield the most effective results in terms of efficacy in a given subject.
  • This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the agent or composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of an agent, population of cells, or composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20 th edition, Williams & Wilkins PA, USA) (2000).
  • these pharmaceutical compositions can be administered by oral administration including sublingual and buccal administration, and parenteral administration including intravenous administration, intramuscular administration, and subcutaneous administration.
  • parenteral administration including intravenous administration, intramuscular administration, and subcutaneous administration.
  • the pharmaceutical composition is administered through an intrathecal or intracranial route of administration.
  • the methods may include administering a therapeutically effective dose of a pharmaceutical composition to the subject.
  • the pharmaceutical composition may include (i) an agent to increase expression or activity of TLX, (ii) an miR-219 inhibitor, or both (i) and (ii).
  • increasing expression or activity of TLX may maintain normal NSC proliferation in the subject.
  • the agent may be a vector expressing a gene encoding TLX.
  • the pharmaceutical composition may further comprise an miR-219 inhibitor. In embodiments where the pharmaceutical composition comprises an miR-219 inhibitor, the pharmaceutical composition may further comprise an agent to increase expression or activity of TLX. In other embodiments where the pharmaceutical composition comprises an agent to increase expression or activity of TLX, the methods may further include administering a therapeutically effective dose of a second pharmaceutical composition to the subject, the pharmaceutical composition comprising an miR-219 inhibitor. In other embodiments where the pharmaceutical composition comprises an miR-219 inhibitor, the methods may further include administering a therapeutically effective dose of a second pharmaceutical composition to the subject, the pharmaceutical composition comprising an agent to increase expression or activity of TLX.
  • the miR-219 inhibitor may be a tough decoy RNA, an RNAi molecule, or an aptamer.
  • the miR-219 inhibitor may be an miR-219-5p hairpin inhibitor or TuD-miR-219.
  • Expression of miR-219 may also be used as a biomarker to detect schizophrenia, bipolar disorder, or depression.
  • the miR-219 may be detected in exosomes or cerebrospinal fluid (CSF).
  • TLX is identified as an upstream regulator of miR-219.
  • p68, Drosha and DGCR8 are identified as novel TLX-interacting molecules, and an unexpected role for TLX in regulating miRNA processing at the post-transcriptional level is uncovered.
  • miR- 219 expression is up-regulated, whereas TLX expression is down-regulated in SCZ NSCs. Overexpression of TLX or an miR-219 inhibitor is able to rescue the proliferative defects in SCZ NSCs.
  • TLX regulates miRNA processing independent of its well-characterized role in transcriptional regulation
  • miR-219 acts downstream of TLX to regulate NSC proliferation and differentiation in mammalian brains.
  • miR-219 expression is elevated, whereas TLX expression is reduced, in D/SC7-mutant SCZ patient iPSC-derived NSCs.
  • Overexpression of TLX or inhibition of miR-219 could rescue the reduced cell proliferation in D/SC7-mutant SCZ NSCs.
  • TLX Disclosed herein is an unexpected role for TLX in miRNA maturation at the post-transcriptional level beyond transcriptional regulation.
  • the RNA helicase p68 a component of the miRNA processing machinery
  • TLX also interacts with p68-associated Drosha and DGCR8, the two main components of miRNA processing machinery. It is shown in this disclosure that TLX inhibits miR-219 processing by interacting with the p68/Drosha/DGCR8 complex, which in turn prevents the miRNA processing machinery from binding to miR-219 primary form.
  • TLX Either knockdown of TLX or blocking the interaction between TLX and the miRNA processing machinery resulted in potent induction of pre-miR- 219 and mature miR-219 expression, but had minimal effect on pri-miR-219 expression.
  • the concept that a transcription factor like TLX can participate in post- transcriptional regulation of gene expression may serve as a general paradigm for many of these classes of cellular factors to control cell fate determination.
  • miR-219 has been shown to induce oligodendrocyte differentiation in electroporated mouse brains that were harvested at E17.5 [27]. However, the induction of oligodendrocyte marker expression in miR-219- electroporated mouse brains harvested at E15.5 was not detected, presumably because brains were harvested at an earlier stage that is active for neurogenesis but not for gliogenesis yet. It is possible that miR-219 could play distinct roles at different developmental stages.
  • miR-219 is dysregulated in neurodevelopmental disorders, including SCZ, bipolar disorder and depression [29,32,42,43]. Understanding the regulation of miR-219 expression in mammalian brains will not only broaden the knowledge about neurodevelopment, but also provide insights into the pathogenesis of neurological disorders. It is shown in this disclosure that TLX represses miR-219 biogenesis in NSCs during mouse brain development. PDGFRa was also identified as a downstream target of the TLX-miR-219 cascade in NSCs. PDGFRa has been shown to be expressed in oligodendrocyte progenitor cells [44] and play a role in oligodendrocyte differentiation downstream of miR-219 [26].
  • DISC1 is required for mouse NSC proliferation [40]. However, little is known about its function in human NSCs. In this disclosure, DISC1 has been found to play a role in regulating human NSC proliferation by studying NSCs derived from D/SC7-mutant SCZ patient iPSCs and genetically engineering isogenic iPSCs with an introduced DISC1 mutation. The observation that miR-219 expression is up- regulated, whereas TLX expression is down-regulated, in D/SC7-mutant NSCs provides a direct link between TLX and miR-219 expression and DISC1 function.
  • TLX KO mice exhibit neuroanatomical and behavioral abnormalities similar to that in D/SC7-mutant mice and SCZ patients, including increased lateral ventricles, reduced cerebral cortex, reduced neurogenesis and memory, and increased anxiety and hyperactivity [2,3,6,7,45-56].
  • the finding of altered expression of TLX in D/SC7-mutant NSCs suggests that mutant DISC1 could regulate TLX expression, which in turn induces abnormal miR-219 expression and inhibition of NSC proliferation.
  • SCZ is a neurodevelopmental disorder for which the pathological mechanism remains elusive. Increasing evidence suggests that miRNAs may play important roles in the etiology of SCZ [57].
  • miRNA-219 is highly up-regulated in the prefrontal cortex of SCZ patients [29,32] and mediates the behavioral effects of the NMDA receptor antagonist Dizocilpine [58]. However, whether miR-219 plays a role in SCZ pathogenesis remained unknown. This disclosure has identified a novel role for miR-219 in SCZ NSCs; elevated miR-219 expression reduces SCZ NSC proliferation.
  • mice Female ICR or Swiss Webster mice at gestation 13.5 were used for in utero electroportion experiments. All mice were produced in the Animal Resource Core of City of Hope. All animal-related work was performed under the IACUC protocol 03038 approved by City of Hope Institutional Animal Care and Use Committee. Mice were maintained in a 12 hr light: 12 hr dark light cycle at 4 mice per cage.
  • Antibodies and immunostaining were used to Flag epitope tag M2 (Sigma, F2426 for IP), HA (1 : 500, Santa Cruz, sc-805), p68 (1 : 1000, Abeam, ab10261 ), Drosha (1 : 1000, cell signaling, #3364), DGCR8 (1 :500, Protein Tech Group, Inc, 10996-1 -AP), BrdU (1 : 5000, Accurate, OBT0030CX), DCX (1 : 300, Santa Cruz, sc-8806) and Ki67 (1 : 200, GeneTex, GTX16667). Immunostaining of embryonic mouse brains was performed using antibodies for DCX and Ki67. For Ki67 staining, antigen retrieval was performed by incubating slides in sodium citrate buffer (10 mM sodium citrate, pH 6.0 and 0.1 % Triton X) at 80°C for 10 min before staining.
  • sodium citrate buffer (10 mM sodium citrate, pH 6.0 and 0.1 % Tri
  • Mouse NSC culture Embryonic mouse NSCs were prepared using an established protocol [63] as follows. E14.5 mouse brains were dissociated by gentle pipetting. The dissociated cells were seeded on polyornithine- and fibronectin-coated plates and cultured in N2 medium (DMEM F12, 25 ⁇ g per ml insulin, 100 ng per ml apo-transferrin, 30 nM sodium selenite, 20 nM progesterone and 100 ⁇ putrescine) supplemented with 10 ng per ml FGF2. Cells were maintained as mycoplasm-free culture as revealed by routine mycoplasm screen using MycoAlert Mycoplasma Detection Kit.
  • N2 medium DMEM F12, 25 ⁇ g per ml insulin, 100 ng per ml apo-transferrin, 30 nM sodium selenite, 20 nM progesterone and 100 ⁇ putrescine
  • NSCs were dissociated into single cells and cultured in N2 medium supplemented with 0.5% fetal bovine serum and 10 ⁇ Forskolin for 5 days.
  • 10 ⁇ BrdU was added to NSCs and pulsed for 30 min. Cells were then fixed and acid treated, followed by immunostaining with anti-BrdU antibody.
  • Transfection of NSCs with reporter plasmid DNA, miRNA or siRNA was performed using TransFectin (BioRad), following manufacturer's instructions.
  • actinomycin D treatment control or TLX siRNA-transduced NSCs were treated with 1 ⁇ actinomycin D for 3 hr, followed by cell harvesting and RNA isolation.
  • DNA fragments containing mouse PDGFRa 3' UTR were subcloned into psiCHECK vector (Promega).
  • the miR-219-5P target site 5'-GACAATCA-3' (SEQ ID NO: 40) in PDGFRa 3' UTR was mutated into 5'-GATCGTCA-3' (SEQ ID NO: 41 ) by site- directed mutagenesis.
  • mice PDGFRa The cDNA of mouse PDGFRa was purchased from ATCC and subcloned into pEF-pUb-RFP vector [4]. To make TLX siRNA or scrambled control RNA-expressing lentiviral vector, DNA fragments containing TLX siRNA or scrambled control siRNA hairpin sequences were subcloned into pHIV-GFP vector [65].
  • DNA fragment containing the Dpi (amino acid residues 341 -359) (SEQ ID NO: 42) or control peptide (amino acid residues 201 -223) (SEQ ID NO: 43) of TLX was fused in frame to three copies of nuclear localization signals and cloned into the CMX-HA or CSC-GFP vector [3].
  • DNA oligos of miR-219 were annealed and cloned into the UEG vector [66].
  • TuD-miR-219 DNA oligos of TuD-miR-219, 5'-TCG AAG AAT TGC GTT CTG ATG GAC AAT CA-3' (SEQ ID NO: 44) and 5'-CTA GTG ATT GTC CAT CAG AAC GCA ATT CT-3' (SEQ I D NO: 45) were annealed and cloned into the U6-TuD vector. The DNA fragment containing the U6 promoter and TuD-miR-219 was then subcloned into pHIV-GFP vector or CMVLV lentiviral vector containing a puromycin-resistant gene [65].
  • 392 bp fragment of pri-miR-219 including the pre-miR-219 hairpin loop was PCR amplified using the following primers: 5 -TTC ATA GAG CTC ACA CCG GCT TGT CCA CCT TAC-3' (SEQ ID NO: 46) and 5'-TTC ATA CTC GAG GAG GAT ACG GAA AGA GGC GAG-3' (SEQ ID NO: 47).
  • the PCR product was digested with Sacl and Xhol site and cloned into the pmirGLO vector (Promega).
  • miR-1224-Glo vector 398 bp fragment of pri-miR-1224 was PCR amplified using the following primers: 5 -GAT AGC TAG CAA TGG CAA CTC CAA GCG TGC T-3' (SEQ ID NO: 48) and 5'-ATG AGG CCG AGG TGG GGC TGA GTC TAG AGA TC-3' (SEQ ID NO: 49).
  • the PCR product was digested with Nhel and Xbal and cloned into the pmirGLO vector (Promega).
  • siRNAs and miRNAs All synthetic siRNAs, miRNAs and their controls were purchased from Dharmacon.
  • miR- 219-5p c-310578-05-0005
  • negative control CN-001000-01 -05
  • miR-219-5p hairpin inhibitor I H-310578-07-0005
  • RNAs from tissue cultured cells or 6 to 8-week-old WT or TLX KO mouse brains were isolated using TRizol (Invitrogen) in accordance with manufacturer's instructions. Oligonucleotides complementary to miRNA sequences were end-labeled with ⁇ 32 ⁇ - ⁇ and used as probes for Northern blot analysis. The sequences for the probes are listed in Figure 16.
  • RT-PCR was performed to detect the levels of primary, precursor and mature miR-219, or TLX and PDGFRa mRNAs.
  • Reverse transcription was performed using Tetro cDNA synthesis kit (Bioline) and the expression levels of pri-miRNA and pre- miRNA of miR-219, and TLX and PDGFRa mRNAs were determined using DyNAmo Flash SYBR Green qPCR mix (Thermoscientific) and StepOnePlus Real-Time PCR system (Applied Biosystems).
  • DyNAmo Flash SYBR Green qPCR mix Thermoscientific
  • StepOnePlus Real-Time PCR system Applied Biosystems
  • TaqMan MicroRNA assay kit was used according to manufacturer's protocol. Data analysis was done by Comparative Ct method. Results were normalized to ⁇ -actin for pri-miRNA, pre-miRNA, TLX and PDGFRa mRNAs, and snoRNA or U6 for mature miRNA.
  • the primers are listed in Figure 16.
  • Extracted peptides were acidified with formic acid (1 %) and injected straightly into the liquid chromatography (LC) mass spectrometry (MS) system, consisting of a binary pump Agilent 1200 HPLC, a 6520 quadrupole time-of-flight mass spectrometer (Agilent), equipped with a chip cube ion source, utilizing a high capacity LC/MS chip (Agilent) with a 150 mm ⁇ 75 ⁇ Zorbax 300SB-C18 on-board analytical reverse phase column and a 160 nl trapping column. 10 ⁇ sized peptide samples were loaded at 4 ⁇ per min.
  • LC liquid chromatography
  • MS mass spectrometry
  • LC was performed with a gradient mobile phase system containing buffer A (0.1 % aqueous formic acid) and B (100% acetonitrile, 0.1 % formic acid).
  • a 50-minute gradient elution from the analytical column was conducted from 7 to 85% buffer B at 300 nl per min.
  • MS and tandem MS analysis of peptide ions with z > 2* was performed in data-dependent mode.
  • Automated collision energy settings were set by the acquisition software, MassHunter (Agilent).
  • the resulting data was analyzed using the GPM X! Tandem search engine (The Global Proteome Machine Organization) with the human protein database and Scaffold (Proteome Software) at a 1 % false discovery rate setting.
  • Lysates were immunoprecipitated using TLX antibody, followed by immunoblotting using indicated antibodies.
  • constructs expressing Dpi TLX residues 341 -359 or a control peptide (TLX residues 201 -223), together with HA-TLX and Flag-Drosha were transfected into HEK293T cells.
  • Cell lysates were immunoprecipitated with Flag antibody (Sigma, F2426), followed by immunoblotting with anti-HA (1 :500, Santa Cruz, sc-805) or anti-Flag antibody (1 :500, Sigma, F1804). Images in Figs. 1 a and 1 b, Figs. 2c, 2d and 2f, and Figs. 5b-5e have been cropped for presentation.
  • RNA immunoprecipitation (RIP). RI P was performed under native condition [68].
  • NSCs were transduced with lentivirus expressing TLX siRNA or scrambled control RNA.
  • Cell pellets were resuspended in ice cold lysis buffer containing 100 mM KCI, 5 mM MgCI2, 10 mM HEPES (pH 7.0), 0.5% NP40, 1 mM DTT, 100 U per ml RNase Inhibitor (Promega), and protease inhibitor cocktail (Roche), and lysates were passed through a 27.5 gauge needle 4 times to promote nuclear lysis.
  • Human iPSC culture and differentiation Human iPSCs were maintained and cultured in Essential 8 (E8) medium (Gibco, A15169-01 ). For NSC differentiation, iPSCs were detached using 0.5 mM EDTA and cultured in E8 medium for 6 days in suspension for embroid body (EB) formation, then switched to neuronal induction medium (50% DMEM/F12, 50% Neurobasal, 0.5 X N2, 0.5 X B27, 2 mM L- Glutamine, 0.1 mM NEAA, and 100 units penicillin/streptomycin) supplemented with 5 ⁇ SB431542 and 0.25 ⁇ LDN for 3 days.
  • E8 Essential 8
  • EB embroid body
  • the EB spheres were transferred into matrigel-coated plates and cultured in neuronal induction medium for 7 days. Rosette structures were mechanically lifted and cultured in neuronal induction medium supplemented with basic FGF (5 ng per ml) and EGF (20 ng per ml) for expansion. Neurospheres were stained for NSC markers using antibodies for SOX1 (1 :500, Millipore, AB15766) and NESTIN (1 : 1000, BD, 61 1659). All the cells used in this study were maintained as mycoplasm-free culture as revealed by routine mycoplasm screen using MycoAlert Mycoplasma Detection Kit.
  • Human NSC proliferation and differentiation Human iPSC-derived NSCs were seeded on matrigel coated 24-well plates in proliferation media and cultured for 24 hr. Lentivirus expressing miR-219 or TuD-miR-219 and a GFP reporter was added to human NSCs in 24-well plates for 16 hr. The virus-transduced cells were labeled by GFP. For proliferation assay, cells were allowed to recover for 2 days and then treated with 10 ⁇ BrdU for 1 hr, followed by immunostaining for BrdU and SOX1 . Nuclei were counter-stained using DAPI .
  • NSC proliferation rate was determined using the percentage of BrdU+SOX1 + cells, which was calculated as BrdU+SOX1 +/DAPI+ cells for non-virus-transduced cells and BrdU+SOX1 +GFP+/GFP+ cells for GFP-expressing virus-transduced cells.
  • NSCs were switched to differentiation medium containing N2 and B27 (1 : 1 ) with 1 ⁇ retinoic acid and 0.5% FBS in DMEM F12 media. Cells were allowed to differentiate for 2 weeks, followed by immunostaining for Tuj1 .
  • the neuronal differentiation rate was determined using the percentage of Tuj1 + cells, which was calculated as Tuj1 +/DAPI+ cells for non-virus-transduced cells and Tuj1 +GFP+/GFP+ cells for GFP-expressing virus-transduced cells.
  • Example 2 TLX represses miR-219 processing
  • pri-miR-219-1 is derived from two primary transcripts, pri-miR-219-1 and pri-miR-219-2. No expression of pri-miR-219-1 was detected in both WT and 7LX KO brains, and not much change in the expression of pri-miR-219-2 was observed in WT and TLX KO brains either (Fig. 1 b & 1 c). Because pri-miR-219-2 was only detected in the brain, pri-miR-219-2 is referred to as pri-miR-219 hereafter.
  • the expression levels were determined for the precursor form of miR- 219 (pre-miR-219) in TLX KO brains.
  • the level of pre-miR-219 increased substantially in TLX KO brains, compared to WT brains, similar to the change in mature miR-219 level, whereas no dramatic change was observed in pri-miR-219 level (Fig. 1 c).
  • the levels of all three forms of miR-219 in TLX knockdown NSCs were examined. Knockdown of TLX by siRNA was confirmed by RT-PCR (Fig. 9).
  • a luciferase- based processing assay was performed.
  • HEK293T cells were transfected with a Iuciferase reporter construct containing pri-miR-219 sequences that include the Drosha/DGCR8 binding sites.
  • the pri-miR-219 sequences were placed between the coding region of the Iuciferase gene and its polyadenylation signal. Cleavage of polyadenylation tails from the Iuciferase transcripts by Drosha/DGCR8 would induce degradation of the Iuciferase transcripts and reduce Iuciferase activity (Fig. 1 e).
  • TLX ectopic expression of TLX in HEK293T cells reduced miR-219 processing, as revealed by increased Iuciferase activity of miR-219-Glo (Fig. 1f).
  • Expression of TLX had no effect on Iuciferase activity of miR-1224-Glo, a reporter that contains part of miR-1224, an miRtron that is processed into pre-miRNA independent of Drosha cleavage 33 (Fig. 1 f).
  • knockdown of TLX in NSCs promoted miR-219 processing, as shown by reduced Iuciferase activity of miR-219-Glo, compared to control RNA-treated cells (Fig.
  • TLX negatively regulates miR-219 processing from the primary form to the precursor form.
  • Example 3 TLX interacts with the miRNA processing machinery
  • RNA helicase p68 is among the proteins that were uniquely represented in the pull-downs of HA-TLX-expressing cells. Seventeen peptides of p68 were detected in the HA immunoprecipitates of HA-TLX-expressing cells, but not in that of control HA-expressing cells.
  • HEK293T cells were transfected with HA-TLX.
  • p68 was detected in the HA-TLX immunocomplex and the interaction was not affected by the treatment with DNase and RNase (Fig. 2c). Because p68 is a component of the Drosha complex that processes pri-miRNAs into pre-miRNAs [18, 19], it was hypothesized that TLX could interact with the miRNA processing machinery via its interaction with p68.
  • HEK293T cells were transfected with Flag-Drosha or Flag- DGCR8 and HA-TLX.
  • HA-TLX was detected in the immunocomplexes of both Flag- Drosha and Flag-DGCR8, independently of DNase and RNase treatment (Fig. 2c).
  • Example 4 miR-219 inhibits mammalian NSC proliferation
  • TLX plays an important role in regulating mammalian NSC proliferation and differentiation [3,4], the observation that TLX regulates miR-219 processing in mouse NSCs led to a hypothesis that miR-219 could be involved in regulating mammalian NSC phenotypes.
  • NSCs were isolated from E14.5 mouse brains and treated with the miR-219 RNA duplex. BrdU labeling was performed to monitor cell proliferation. Treatment with miR-219 reduced cell proliferation substantially (Fig. 3a, c), but had minimal cytotoxicity (Fig. 10a). These results indicate that miR-219 inhibits mammalian NSC proliferation. The effect of miR-219 was then tested on NSC differentiation.
  • E14.5 mouse NSCs were treated with the miR-219 RNA duplex and cultured in differentiation medium.
  • Treatment with miR-219 increased the percentage of ⁇ tubulin (Tujl )-positive neurons substantially, compared to treatment with a control RNA (Fig. 3b, d). These results indicate that miR-219 promotes mammalian NSC differentiation into neurons.
  • miR-219 RNA duplex was electroporated together with an RFP-expressing vector into NSCs of E13.5 embryonic brains in uterus. The brains were dissected at E15.5 and analyzed by immunohistochemistry. Immunostaining with Ki67, a proliferation marker, revealed that overexpression of miR-219 decreased cell proliferation in the ventricular zone and subventricular zone (VZ/SVZ) of mouse brains, where NSCs reside (Fig. 3e, f). To determine the effect of miR-219 overexpression on neuronal differentiation, immunostaining with doublecortin (DCX), a neuronal marker, was performed.
  • DCX doublecortin
  • miR-219 has been shown to induce oligodendrocyte differentiation in in utero electroporated mouse brains harvested at E17.5 [27]. However, the induction of either astrocyte marker GFAP or oligodendrocyte marker MBP expression was not detected in miR-219-electroporated brains harvested at E15.5 (Figs. 10b and 10c), presumably because brains were analyzed at an early cortical developmental stage when neurogenesis is active but gliogenesis is not yet. These results indicate that miR-219 inhibits mammalian NSC proliferation and promotes their neuronal differentiation during early brain development.
  • Example 5 miR-219 acts downstream of TLX to regulate NSC phenotypes
  • TargetScan which revealed a set of candidate miR-219 targets, the 3' UTR of which can base pair with miR-219.
  • PDGFRa is a confirmed miR-219 target [26] and is expressed in NSCs [34].
  • RT-PCR showed that the expression of PDGFRa was dramatically decreased in TLX KO brains (Fig. 1 1 a).
  • miR- 219 repressed the activity of the luciferase reporter with the wild type PDGFRa 3' UTR, but not the reporter that with the mutant 3' UTR, in which the base pairing with miR-219 was destroyed (Fig. 1 1 b), suggesting that miR-219 inhibits PDGFRa expression.
  • PDGFRa regulates NSC proliferation and differentiation in vivo was studied. In utero electroporation of PDGFRa siRNA into E13.5 mouse brains reduced NSC proliferation as shown by decreased Ki67+RFP+ cells in the VZ/SVZ, whereas the number of RFP+ cells migrated to the CP was increased (Figs. 12a-d). To determine if PDGFRa is a critical downstream target gene of miR-219 in NSC regulation, if the effect of miR-219 on NSC proliferation and differentiation could be reversed by overexpressing PDGFRa was tested.
  • Example 7 A TLX peptide promotes miR-219 processing
  • TLX residues 340-359 were critical for the TLX-p68 interaction (Fig. 5a, b).
  • Deletion of TLX residues 340-359 also reduced the interaction of TLX with Drosha dramatically (Fig. 5a, c).
  • the TLX region spanning residues 340 to 359 was determined to be the Drosha/p68-interaction (Dpi) domain (Fig. 5a).
  • TLX-Drosha an HA-tagged TLX peptide containing the Dpi domain (Dpi) was co- expressed with HA-tagged full-length TLX (HA-TLX) and Flag-Drosha in HEK293T cells.
  • Dpi was expressed together with HA-TLX and Flag-HDAC5, a known transcriptional corepressor of TLX [1 1 ].
  • HA-TLX and Flag-HDAC5 a known transcriptional corepressor of TLX [1 1 ].
  • the interaction of TLX with HDAC5 was not blocked by Dpi (Fig. 5e).
  • the Dpi peptide was electroporated into NSCs and determined miR-219 processing by evaluating the levels of the three forms of miR-219, pri-miR-219, pre- miR-219 and mature miR-219.
  • the levels of both pre-miR-219 and mature miR-219 forms increased considerably, whereas no significant change was detected in the level of pri-miR-219 (Figs. 5g-5i).
  • NSCs from E14.5 mouse brains were transduced with lentivirus expressing Dpi or a control peptide and a GFP reporter. Compared to the control peptide, expression of Dpi reduced cell proliferation substantially (Figs. 6a and 6b).
  • NSCs were co-transduced with Dpi and the miR-219 decoy inhibitor, TuD-miR- 219. Expressing TuD-miR-219 rescued the Dpi-mediated inhibition of NSC proliferation substantially (Figs.
  • Example 8 Elevated miR-219 expression inhibits SCZ NSC proliferation
  • Wild type (WT) NSCs derived from iPSCs of unaffected individuals were used as controls [37,38]. Both SCZ and WT iPSC- derived NSCs expressed human NSC markers SOX1 and NESTIN (Fig. 7b).
  • RT-PCR revealed that the level of miR-219 increased substantially in D/SCi-mutant SCZ NSCs, compared to WT control NSCs (Fig. 7c).
  • the isogenic iPSC lines C1 M and C3M were used, in which the 4 bp deletion seen in the SCZ patients was introduced into the DISC1 gene in the WT control iPSC lines C1 and C3 [38]. Similar to what was seen in the SCZ NSCs, a considerable increase was observed in miR-219 level in C1 M and C3M NSCs, compared to that in their isogenic WT controls (Fig. 7c).
  • miR-219 was overexpressed in WT NSCs using an miR-219-expressing retroviral vector and NSC proliferation was determined by BrdU and SOX1 double labeling. Reduced cell proliferation was observed in miR-219- overexpressing WT NSCs compared to control vector-expressing WT NSCs, in a manner similar to the reduced cell proliferation observed in D/SC7-mutant NSCs when compared to WT NSCs (Fig. 8a). In parallel, the miR-219-overexpressing WT NSCs were induced for neuronal differentiation. The rate of neuronal differentiation was determined by the percentage of Tuj1 + cells.
  • D/SC7-mutant NSCs To determine if the proliferative defect in D/SC7-mutant NSCs indeed resulted from abnormally elevated miR-219 expression, miR-219 in D/SC7-mutant NSCs was inhibited using TuD-miR-219. NSC proliferation was monitored by BrdU and SOX1 double labeling. Treating the D/SC7-mutant NSCs (D1 , D2, C1 M and C3M) with TuD-miR-219 increased the proliferative rate in these cells substantially, largely rescuing the proliferative defects of the D/SC7-mutant NSCs (Fig. 8b).
  • TuD-miR-219 Treatment with TuD-miR-219 also reversed the elevated differentiation in DISC1- mutant NSCs considerably, as revealed by the reduced percentage of Tuj1 + cells, compared to that in cells treated with a control vector (Fig. 15b). These results indicate that miR-219 plays an important role in regulating cell proliferation in DISC1- mutant SCZ NSCs and that an miR-219 inhibitor could rescue the proliferative defect in these cells.
  • Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain. Mol Endocrinol 22, 56-64 (2008).
  • Histone demethylase LSD1 regulates neural stem cell proliferation. Mol Cell Biol 30, 1997-2005 (2010). Yokoyama, A., Takezawa, S., Jr, R., Kitagawa, H., and Kato, S.
  • PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling. Neuron 51 , 187-199 (2006). Haraguchi, T., Ozaki, Y., and Iba, H. Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in
  • Margolis, R. L A frameshift mutation in Disrupted in Schizophrenia 1 in an American family with schizophrenia and schizoaffective disorder. Mol Psychiatry 10, 758-764 (2005). Chiang, C. H., Su, Y., Wen, Z., Yoritomo, N., Ross, C. A., Margolis, R. L, Song, H., and Ming, G. L. Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation. Mol Psychiatry 16, 358-360 (201 1 ). Wen, Z., Nguyen, H. N., Guo, Z., Lalli, M.
  • M. Fierce a new mouse deletion of Nr2e1 ; violent behaviour and ocular abnormalities are background-dependent. Behav Brain Res 132, 145- 158 (2002). Clapcote, S. J., Lipina, T. V., Millar, J. K., Mackie, S., Christie, S., Ogawa, F., Lerch, J. P., Trimble, K., Uchiyama, M. , Sakuraba, Y., Kaneda, H., Shiroishi, T., Houslay, M. D., Henkelman, R. M., Sled, J. G., Gondo, Y., Porteous, D. J., and Roder, J. C.
  • MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction.
  • Disruption of neurogenesis on gestational day 17 in the rat causes behavioral changes relevant to positive and negative schizophrenia symptoms and alters amphetamine-induced dopamine release in nucleus accumbens.

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Abstract

La présente invention concerne des méthodes de traitement de troubles neurodéveloppementaux tels que la schizophrénie (SCZ), un trouble bipolaire ou la dépression. Les méthodes impliquent l'inhibition de l'expression de miR-219 ou la surexpression de TLX, favorisant ainsi la prolifération de cellules souches neurales (NSC) chez les sujets.
PCT/US2017/021962 2016-03-10 2017-03-10 Tlx et mir-219 utilisés comme cibles thérapeutiques potentielles pour des troubles neurodéveloppementaux WO2017156491A1 (fr)

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